DIAGNOSTIC MICROBIOLOGY

Transcription

1 DIAGNOSTIC MICROBIOLOGY Libuše Kolářová Václava Adámková Institute for Immunology and Microbiology, 1st. Fac.Med.,Charles Univ. in Prague Czech Republic

2 Which factors should precipitate testing? CLINICAL SYMPTOMS CONTACT WITH INFECTED INDIVIDUALS TRAVEL HISTORY IMMUNE STATUS OF THE PATIENT (e.g. compromised patient -increase in the number of patients whose immune systems are compromised through underlying illness, chemotherapy, transplantation) DOCUMENTED PREVIOUS INFECTION SCREENING (e.g., outbreak situation)

3 How determine causative agent of the disease? DIRECT OR INDIRECT METHODS Direct methods (e.g, microscopy, cultivation of specific nucleid acids, detection of specific antigens) = highly specific and unambigously recommendable, however, in some cases: either low sensitivity (microscopy) or expensive, but important - the possibility of testing the sensitivity to ATB. Indirect methods (e.g. serological methods = sometimes can be of low sensitivity and specificity)

4 How determine causative agent of the disease? Examination of exact sample (dependence on clinical symptoms and signs!!!) isolated: from exact site; at the exact time interval; transport to laboratory examination under adequate conditions (standards) examined by adequate methods (standards)

5 MATERIAL Clinical symptoms = specific material in which the causative agent can be detected e.g., stool urine blood cerebrospinal fluid sputum organ biopsies, aspirates smears, etc. = isolation at exact time

6 Body site Specimen (examples) Test options (examples) Blood whole blood, serum, anticoagulated blood, etc. culture, QBC microhematocrit centruifugation, Buffy coat films, Knott concentration, membrane filtr. techniq. immunoassays, animal inoculation Bone narrow aspirate culture, histopathology, thick and thin smears, PCR CNS spinal fluid, brain biopsy specimen culture, wet examination, stained smears, immunoassays, PCR Eye apirates from below surface, biopsy specimen culture, wet preparation, stained smears, Skin smears, scrapings, apirates from below surface, biopsy specimen culture, histopathologic testing, squash preps (stained smears), Intestinal tract fresh stool culture,direct wet smear, concentr., permanent stained smear, ag. det. Liver and spleen anal smear preserved stool sigmoidoscopy material duodenal contents anal impression smear sputum, induced sputum, broncholaveolar lavage fluid, transbronchial aspirate, brush biopsy specimen, aspirate, open-lung biopsy specimen culture, direct wet smear concentration, permanent stained smear direct wet smear, stained smear exam. of tapes for pinworm eggs wet preparation, stained smear, immunoassays, histopathologic testing, PCR Lymph node biopsy specimen culture, stained smear, histopathol. test., PCR Muscle biopsy specimen histopathologic testing, PCR Urogenital system vaginal discharge, urethral discharge, prostatic secretions, urine, biopsy specimen culture, wet preparation, stain.smears, histopathol.test.

7 DETECTION OF THE AGENT 1) DIRECT macroscopically or microscopically culture: predermined culture media or tussue cultures under controlled laboratory conditions non-concentration methods: nativ fresh mounts stained smears concentration methods: flotation sedimentation filtration specific methods: detection of DNA, circulating antigens Detection of the parasite DNA: limited use Material: e.g. incondesable blood, stool, urine (fresh, frozen, fixed in pure 100% alcohol)

8 MACROSCOPICAL examination of samples Cestoda: proglotides Ascaris lumbricoides

9 CULTURE

10 BLOOD CULTURE Very important - e.g., due to sepsis, pneumonia, fever of unknown origin, puerperal sepsis, pelvic inflammation, neonatal epiglottitis Source: Wikimedia Commons

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15 CEREBROSPINAL FLUID CULTURE + other normally sterile fluids e.g., peritoneal, pleural, synovial 1-3 ml of fluid transported to the laboratory as soon soon as possible Source: Wikimedia Commons

16 DEFINITION OF SIGNIFICANT BACTERIURIA IN PREGNANCY in an asymptomatic pregnant woman, bacteriuria is considered significant if two consecutive voided urine specimens grow > 10 5 cfu/ml of the same bacterial species on quantitative culture; or a single catheterised specimen grows > 10 5 cfu/ml of a uropathogen in a pregnant woman with symptoms compatible with UTI, bacteriuria is considered significant if a voided or catheterised urine specimen grows > 10 3 cfu/ml of a uropathogen

17 > 10 3 cfu/ml of uropathogens in a mid-stream sample of urine (MSU) in acute uncomplicated cystitis in women > 10 4 cfu/ml of uropathogens in an MSU in acute uncomplicated pyelonephritis in women. > 10 5 cfu/ml of uropathogens in an MSU in women, or > 10 4 cfu/ml uropathogens in an MSU in men, or in straight catheter urine in women, in a complicated UTI. In a suprapubic bladder puncture specimen, any count of bacteria is relevant.

18 Escherichia coli 10 8 / ml

19 ASYMPTOMATIC BACTERIURIA diagnosed if two cultures of the same bacterial strain (in most cases the species only is available), taken > 24 h apart, show bacteriuria of > 10 5 cfu/ml of uropathogens

20 MICROSCOPICAL examination of samples Entamoeba histolytica cysts Plasmodium falciparum

21 CULTURE & MICROSCOPY Material: Smears e.g., of vaginal mucosa Trichomonas vaginalis 1. MICROSCOPY EXAMINATION of smear - in vivo, staining by Giemsa - MOP) 2. CULTURE MIKROSCOPY 21

22 MICROSCOPY NATIVE WET MOUNTS Enterobius vermicularis

23 MICROSCOPY WET FRESH STAINED MOUNT Staining e.g. by Lugol, s iodine (e.g.,amoebae)

24 MICROSCOPY WET FRESH MOUNTS OF ORGAN BIOPSIES Quantitative compressed biopsy technique (QCTB) e.g. Schistosoma eggs

25 MICROSCOPY STAINED DRY SMEARS e.g. blood: malaria, filariases material: peripheral blood Thick smear stained by Giemsa (no fixation by methanol) Source: Wikimedia commons Thin smear: following fixation by methanol, staining by Giemsa

26 PERIPHERAL BLOOD: THICK SMEARS

27 PERIPHERAL BLOOD: THIN SMEARS

28 MICROSCOPY EXAMINATION OF FAECAL SMEARS smear fixation staining e.g. eggs of intestinal protozoa

29 MICROSCOPY EXAMINATION OF FAECAL THICK SMEARS Kato-Katz Technique celophane faecal thick smears (glycerol-malachite green or glycerol-methylene blue solution; solutions are poured into the cellophane strips and soaked in this solution in a jar) e.g. eggs of intestinal helmints

30 MICROSCOPY EXAMINATION OF CONCENTRATED MOUNTS FECAL CONCENTRATION PROCEDURES various layers seen in the tubes after centrufugation (the surface film should be within 2 to 3 mm of the tube rim) A) Formalin-ether B) Zinc sulfate

31 MICROSCOPY EXAMINATION OF CONCENTRATED MOUNTS FLOTATION Zinc Sulfate (33% aqueous solution) e.g. protozoan cysts, helminthic eggs

32 DETECTION OF THE AGENT 2) INDIRECT using specifical methods, detection of specific antibodies in the serum, vitreous humour, CTF(when the agent is losalised in the organ/tissue) methods: e.g., ELISA, IHA, IFAT, WB material: condensable blood

33 Examples

34 Trichomonas vaginalis Trophozites: vaginal cavity and urethra Transmission: veneral contacts Diagnose: examination of discharge,vaginal smears (staining mounts, culturing) 1) FRESH MOUNTS (culture) fa: anterior flagella, fr: posterior flagella, n: nucleus, ax: axostyle, um: undulating membrane size: µm x 6-20 µm

35 STAINED DRY SMEARS

36 Plasmodium Disease: malaria Transmission: vector Diagnose: examination of peripheral blood smears and other techniques such as PCR 3) STAINED BLOOD SMEARS Thick smear Thin smear

37 a) Entamoeba histolytica b) Giardia intestinalis Disease: a),b) intestinal and a) extraintestinal infections Transmission: per os (food born infection) Diagnose: intestinal: examination of stool, extraintestinal: detection of antibodies, immaging 4,5) STAINED FAECAL SMEARS Giardia Entamoeba histolytica

38 Ascaris lumbricoides Disease: mainly intestinal infection Transmission: per os (food born infection) Diagnose: examination of stool 6) MOUNTS PREPARED BY FLOTATION METHOD fertile infertile Size: 60 x 45 µm Size: 80 x 45 µm

39 Schistosoma mansoni Disease: intestinal and organ infection Transmission: by cercariae (water-born infection) Laboratory dg.: examination of stool, detection of antibodies, immaging 7) Quantitative compressed biopsy technique Size: x µm

40 Thank you for attention